The Society of Nematologists 2011. Mustard seed meal …€¦ · Mustard seed meal mixtures: management of Meloidogyne incognita on pepper and potential phytotoxicity SUSAN L. F

Journal of Nematology 43(1):7–15. 2011.� The Society of Nematologists 2011.

Mustard seed meal mixtures: management of Meloidogyne incognitaon pepper and potential phytotoxicity

SUSAN L. F. MEYER,1 INGA A. ZASADA,2 SAMUEL B. ORISAJO,3 MATTHEW J. MORRA4

Abstract: Meals produced when oil is extracted from seeds in the Brassicaceae have been shown to suppress weeds and soilbornepathogens. These seed meals are commonly used individually as soil amendments; the goal of this research was to evaluate seed mealmixes of Brassica juncea (Bj) and Sinapis alba (Sa) against Meloidogyne incognita. Seed meals from Bj ‘Pacific Gold’ and Sa ‘IdaGold’were tested alone and in combinations to determine rates and application times that would suppress M. incognita on pepper(Capsicum annuum) without phytotoxicity. Rates of soil application (% w/w) for the phytotoxicity study were: 0.5 Sa, 0.2 Bj, 0.25 Sa +0.25 Bj, 0.375 Sa + 0.125 Bj, 0.125 Sa + 0.375 Bj, and 0, applied 0 – 5 weeks before transplant. Overall, 0.2% Bj was the least toxic mealto pepper seedlings. By comparison, 0.5% S. alba seed meal did not reduce lettuce (Lactuca sativa) seed germination at week 0, but allseed meal treatments containing B. juncea prevented or significantly reduced germination at week 0. The seed meals did not affectlettuce seed germination at weeks 1-5, but hypocotyl growth was reduced by all except 0.2% Bj at weeks 1, 4 and 5. Brassica juncea andSa meals were tested for M. incognita suppression at 0.2, 0.15, 0.1 and 0.05%; mixtures were 0.1% Sa + 0.1% Bj, 0.15% Sa + 0.05% Bj,and 0.05% Sa + 0.15% Bj. All treatments were applied 2 weeks before transplant. The 0.2% Bj and 0.05% Sa + 0.15% Bj treatmentsoverall had the longest shoots and highest fresh weights. Eggs per g root were lowest with 0.1 – 0.2% Bj amendments and the seedmeal mixtures. The results indicate that Bj and some Bj + Sa mixtures can be applied close to transplant to suppress M. incognitapopulations on pepper; consequently, a seed meal mixture could be selected to provide activity against more than one pest orpathogen. For pepper, care should be taken in formulating mixtures so that Sa rates are low compared to Bj.

Key words: amendment, biofuel byproducts, Brassica, glucosinolate, management, Meloidogyne incognita, mustard seed meal, root-knot nematode, Sinapis.

Mustard seed meals are byproducts resulting fromcrushing seed to provide oil for the production ofbiodiesel. Because members of the Brassicaceae areplanted as crops that can be incorporated into soil asgreen manures to suppress plant diseases, mustard seedmeals have also been studied as soil amendments formanagement of weeds and soilborne pathogens (Brownand Morra, 1995, 2005; Cohen et al., 2005; Mazzola andMullinix, 2005; Cohen and Mazzola, 2006; Vaughn et al.,2006; Mazzola et al., 2007, 2009; Rice et al., 2007; Boydstonet al., 2008; Hoagland et al., 2008). This agricultural useof seed meals increases the economic viability of bio-diesel production by providing an application for themeal byproduct, and supplies growers with a manage-ment tool for pests and pathogens (Cohen and Mazzola,2004; Brown and Morra, 2005).

Mustard seed meals have also been investigated formanagement of plant-parasitic nematodes. Plant-parasiticnematodes suppressed by soil amendment with mus-tard seed meals include genera such as Belonolaimus(Cox et al., 2006), Meloidogyne (Rahman and Somers,2005; Henderson et al., 2009; Lazzeri et al., 2009; Zasadaet al., 2009), Pratylenchus (Mazzola et al., 2007, 2009; Yuet al., 2007; Walters et al., 2009; Zasada et al., 2009) and

Tylenchulus (Walker, 1997). The biocontrol activity of thebeneficial nematode Steinernema was also disrupted byapplication of mustard seed meals (Henderson et al.,2009). Possible mechanisms of action include productionof toxins upon breakdown of glucosinolates (Lazzeriet al., 1993, 2004; Donkin et al., 1995; Buskov et al., 2002;Zasada and Ferris, 2003; Yu et al., 2005; Zasada et al.,2009), alteration in the bacterial community, and/orinduction of plant systemic resistance by production ofnitric oxide by soil bacteria (Mazzola et al., 2001, 2007;Cohen et al., 2005; Cohen and Mazzola, 2006).

Two of the mustard seed meals that were activeagainst nematodes in soil tests were Brassica juncea andSinapis alba. Of these two meals, B. juncea seed mealexhibited higher nematotoxicity (Mazzola et al., 2009;Zasada et al., 2009). For example, rates of 2.5% and10% S. alba (w/w in dry soil) seed meal were requiredfor 100% suppression of Meloidogyne incognita and Pra-tylenchus penetrans, respectively, but only 0.5% B. junceawas needed for 100% suppression of both nematodes(Zasada et al., 2009). Efficacy of S. alba seed mealagainst nematodes was increased when the seed mealwas ground to a small particle size; ground S. alba sup-pressed P. penetrans populations by 93%, compared to37 – 46% suppression with the same seed meal appliedas a pellet (Zasada et al., 2009).

Because chemistry varies with type of seed meal, thepotential exists for a combination of B. juncea andS. alba to provide enhanced activity against pathogensand pests compared to seed meals applied alone. How-ever, any seed meal combination needs to be selected tooptimize nematicidal activity while minimizing phyto-toxicity to the crop plant. The current study was con-ducted to compare the efficacy of individual seed mealsand seed meal combinations for suppression of M. in-cognita on pepper (Capsicum annuum). The specific

Received for publication September 30, 2010.1USDA, ARS, Nematology Laboratory, Henry A. Wallace Beltsville Agricul-

tural Research Center (BARC)-West, Bldg. 010A, Rm. 112B, 10300 BaltimoreAve., Beltsville, MD 20705-2350, USA.

2USDA-ARS Horticultural Crops Research Laboratory, 3420 NW OrchardAve., Corvallis, OR 97330, USA.

3Plant Pathology Section, Crop Protection Division, Cocoa Research Instituteof Nigeria, P.M.B. 5244, Ibadan, Oyo State, Nigeria.

4Soil & Land Resources Division, PO Box 442339, University of Idaho, Moscow,ID 83844-2339, USA.

Mention of trade names or commercial products in this publication is solelyfor the purpose of providing specific information and does not imply recom-mendation or endorsement by the U.S. Department of Agriculture.

E-mail: [email protected] paper was edited by Kris Lambert.

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objectives of this study were to: 1) determine phyto-toxicity of seed meals and seed meal mixtures to pepperseedlings and to lettuce (Lactuca sativa) seeds (the lat-ter was included because lettuce seed bioassays area standard means of testing for phytotoxicity); 2) de-termine application rates and times for treatments withB. juncea ‘Pacific Gold’ and S. alba ‘IdaGold’ seed mealsthat would suppress M. incognita on pepper withoutphytotoxicity; and 3) investigate whether nematode-suppressive activity could be retained or improved bymixing the two seed meals.

MATERIALS AND METHODS

Meloidogyne incognita inoculum: Inoculum of M. in-cognita Race 1, originally isolated in MD, was grown onpepper ‘PA-136’ in greenhouse pots. Eggs for green-house experiments were obtained from the roots of3-month-old pepper plants. Roots were rinsed and thenimmersed in 0.6% sodium hypochlorite for 1 min torelease eggs from egg masses. The eggs were collectedon a sieve following sugar centrifugation, rinsed inwater, stored overnight at 4 8C and used the next day(Meyer et al., 2008).

Phytotoxicity of seed meals to pepper seedlings and lettuceseeds: The two seed meals tested were Sinapis alba ‘IdaGold’and Brassica juncea ‘Pacific Gold.’ Seed meal treatmentswere placed into pots at week 0 (pepper seedling trans-plant day) and weeks 1, 2, 3, 4 and 5 prior to pepperseedling transplant (week 1 was 7 and 5 days prior topepper transplant in Trial 1 and Trial 2, respectively).Steamed, air-dried soil (loamy sand; 16 sand:9 compost(volume:volume); 83.1% sand, 6.4% silt, 10.5% clay; pH6.9; 0.8% organic matter) was placed into 16.5 x 14.9cm sealable plastic bags, and seed meal that had beenbroken into smaller flakes with a mortar and pestle(< 7 mm and > 0.8 mm; most of the seed meal passedthrough a 2 mm mesh) was added at the appropriaterate for each treatment. The combined total weight ofsoil and mustard seed meal was 400 g per bag. Rates ofmustard seed meal application (dry weight meal to dryweight soil) are listed in Table 1. The soil and seed mealwere mixed, each bag received 48 mL water (70% waterholding capacity of the soil), and the amended andnonamended soils were placed into 10-cm-diameterpots. Pots were watered 1–2 times per day, and 6-week-old pepper (‘PA 136’) seedlings that had been plantedin starter mix (Premier Pro-mix�, Premier HorticultureInc., Quakertown, PA) were transplanted into alltreatments on the same day. The pots were arranged ina randomized complete block design and harvested 12days later. The greenhouse was maintained at 24 – 29 8Cand natural and supplemental lighting were combinedfor a 16-h daylength. At harvest, the number of viableplants, shoot lengths (from soil to growing tip) andshoot and root fresh weights were recorded. The ex-periment was conducted twice, with five seedlings per

seed meal treatment/application time combination ineach of the two trials; n = 10.

For lettuce seed germination trials, 20 g of soil wereremoved from each of three pots/treatment and placedon filter paper in a Petri dish. The next day, 8 ml waterand lettuce seeds (10 or 20) were added to each Petridish. The dishes were placed at a 458 angle and in-cubated for 5 days at 26 8C. After 5 days, number ofseeds germinated, hypocotyl length, and root lengthwere measured from 10 seeds per Petri dish. The ex-periment was conducted twice; n = 60.

Suppression of M. incognita on pepper by seed meals. Basedon the results of the phytotoxicity tests, 2 weeks wasselected as the time for seed meal application prior totransplanting, and individual seed meals were appliedat rates # 0.2%. All seed meal mixtures were applied fora combined total of 0.2%. The seed meal treatments arelisted in Table 1.

Greenhouse conditions for the nematode suppres-sion studies were as listed above. Mustard seed mealtreatments and steamed, air-dried soil (loamy sand; 16sand:9 compost; 82.9% sand, 5.3% silt, 11.8% clay; pH7.3; 0.8% organic matter) were mixed in plastic bags.The combined total weight of soil and mustard seedmeal was 400 g per bag. Each M. incognita treatmentreceived 5,000 eggs (collected as described above) in5 ml water per bag, plus an additional 43 ml water. Thecontrol without nematodes received 48 ml water perbag. The eggs and/or water were mixed into the soil/mustard seed meal mixtures and in nonamended soil,and then the soils were placed into 10-cm-diameter pots

TABLE 1. Application rates of Sinapis alba and Brassica juncea seedmeals tested for phytotoxicity and for suppression of Meloidogyneincognita populations.

Phytotoxicity test with pepperand lettucea (% seed meal

weight to weight soil)

M. incognita suppression onpepperb (% seed mealweight to weight soil)

Single meal amendments0.5% S. alba0.2% B. juncea 0.2% S. alba

0.2% B. juncea0.15% S. alba0.15% B. juncea0.1% S. alba0.1% B. juncea0.05% S. alba0.05% B. juncea

Meal combination amendments0.25% S. alba + 0.25% B. juncea

0.1% S. alba + 0.1% B. juncea0.375% S. alba + 0.125% B. juncea

0.15% S. alba + 0.05% B. juncea0.125% S. alba + 0.375% B. juncea

0.05% S. alba + 0.15% B. junceaNo mealNonamended Nonamended 6 M. incognita

aThe phytotoxicity test was conducted with pepper seedlings and with ger-minating lettuce seeds.

bSuppression of M. incognita was studied on pepper plants in a separate ex-periment from the phytotoxicity test conducted with lettuce and pepper.

8 Journal of Nematology, Volume 43, No. 1, March 2011

in the greenhouse. The treatments were watered 1–2times per day, as needed. Two weeks later, one 6-week-old pepper seedling that had been planted in PremierPro-mix� starter mix was transplanted into each pot.After transplant, the pots were arranged in a random-ized complete block design. Plants were fertilized asneeded with Osmocote� Plus 15N-9P-12K fertilizer(Scotts-Sierra Horticultural Products Co., Marysville,OH). Plants were harvested 5 weeks after transplant. Atharvest, shoot lengths and fresh weights were recordedas described above. Eight pots were used per treatmentin each trial, and the experiment was conducted twice.

Following harvest, roots were removed from pots andsoil was rinsed from the roots. Root fresh weights wererecorded. Total numbers of galls per root system werecounted and root galling index values were assessed asfollows (Daulton, 1959): 0 = free from galls; 1 = less thanfive galls; 5 = trace to 25 galls, 10 = 26 to 100 galls; 23 =more than 100 galls. Roots were stored at 4 8C until eggswere extracted and counted. To extract eggs, roots werecut into pieces and blended on low speed in 0.6% sodiumhypochlorite for 1 min, and then poured onto nested 60 /500 (250 mm / 25 mm) mesh sieves. The eggs were rinsedwith water, collected from the 500-mesh sieve and storedat 4 8C until counting.

Statistical methods: For the pepper seedling phytotox-icity study, the characteristics analyzed were numbers oflive plants, shoot lengths, and shoot and root freshweights. Shoot length and shoot and root fresh weightwere log10(x+1)-transformed to meet the assumptionsof analysis of variance (ANOVA). For the lettuce seedphytotoxicity study, the characters of interest were seedgermination, hypocotyl length, and root length. Thedata were not log transformed for analysis. To de-termine effects of seed meals on pepper plants andsuppression of M. incognita, data analyzed were shootlengths, shoot and root fresh weights, root gall indices,and number of M. incognita eggs/g root. The data werenot log transformed prior to analysis. Data were ana-lyzed with the statistical package JMP (SAS Institute,Cary, NC). Differences among treatments were de-termined by ANOVA, and means were compared usingTukey Kramer’s adjustment for multiple comparisons(P # 0.05). Data presented are nontransformed means,with ± standard errors (SE) in the figures.

RESULTS

Phytotoxicity of seed meals to pepper seedlings and lettuceseeds: Pepper seedling viability. Viability in the non-amended control (0% seed meal) was 100% at alltransplant times (Table 2). At week 0 all pepper seed-lings transplanted into mustard seed meals died, re-gardless of rate or combination. At week 1, 0.5% S. albaseed meal resulted in death of all pepper seedlings, andthe two seed meal mixtures containing the higher ratesof S. alba, 0.25 and 0.375%, also caused death of most of

the seedlings (10% to 30% viable, respectively). Treat-ment with B. juncea seed meal alone or with the lowS. alba + high B. juncea (0.125% + 0.375%) mixture wasnot toxic to pepper transplanted one week after appli-cation. When pepper was transplanted at weeks 2 and 3,only 0.5% S. alba seed meal caused significant seedlingdeath compared to the control. By week 5, only 0.5% S.alba and 0.375% S. alba + 0.125% B. juncea resulted inless than 100% viability, although this was not signifi-cantly lower than the control. Plant viability in all othertreatments was 100%.

Pepper seedling shoot length and root and shootfresh weight. At weeks 1-5, the control and 0.2% B. junceaseed meal had the longest pepper seedling shoots

TABLE 2. Viability of pepper seedlings and germination of lettuceseeds in soil amended with Sinapis alba and Brassica juncea seed meals.

Rates (% w/w)a Weekb

Viable pepperseedlings

(%)c

Germinatedlettuce

seeds (%)

0.5% Sinapis alba 0 0 d 67 a1 0 d 69 a2 30 bcd 76 a3 30 bcd 65 a4 80 ab 77 a5 80 ab 80 a

0.2% Brassica juncea 0 0 d 5 b1 100 a 83 a2 100 a 86 a3 100 a 78 a4 100 a 78 a5 100 a 77 a

0.25% S. alba +0.25% B. juncea 0 0 d 0 b1 10 cd 73 a2 70 ab 83 a3 70 ab 68 a4 70 ab 68 a5 100 a 78 a

0.375% S. alba +0.125% B. juncea 0 0 d 21 b1 30 bcd 83 a2 60 abc 80 a3 60 abc 74 a4 80 ab 78 a5 70 ab 65 a

0.125% S. alba +0.375% B. juncea 0 0 d 0 b1 90 a 84 a2 80 ab 77 a3 100 a 78 a4 100 a 78 a5 100 a 82 a

Nonamended control 0 100 a 72 a1 100 a 84 a2 100 a 90 a3 100 a 88 a4 100 a 88 a5 100 a 83 a

aAmended and nonamended soils were placed into pots in the greenhouse0 to 5 weeks prior to pepper seedling transplant and to lettuce seed germina-tion in Petri dishes.

bWeek 0 = pepper seedlings transplanted the day that amended and non-amended soil was placed into pots.

cValues are the means from two trials, with five replicates of each treatmentper trial for pepper seedlings (n = 10), and thirty replicates of each treatmentper trial for lettuce seeds (n = 60). Within a column, values followed by the sameletter are not significantly different (P < 0.05) according to Tukey’s adjustmentfor multiple comparisons. Significance letters are not comparable betweencolumns.

Mustard meal phytotoxicity and nematotoxicity: Meyer et al. 9

(Fig. 1A) and the highest root fresh weights (Fig. 1B).Sinapis alba seed meal at 0.5% had the most phytotoxiceffect on pepper shoot lengths and root fresh weights(Figs. 1A, 1B). Treatment with the three seed mealmixtures resulted in a trend toward intermediate shootlengths, and some phytotoxicity on roots.

Shoot fresh weights showed a trend similar to that ofshoot lengths (data not shown). To summarize briefly,the control and 0.2% B. juncea seed meal had thegreatest shoot fresh weights. Low shoot fresh weightswere recorded from the 0.5% S. alba seed meal at 3, 4and 5 weeks, and from a few other treatments applied 1and 2 weeks prior to transplant. The control and 0.2%B. juncea seed meal at 1 and 2 weeks were the onlytreatments that resulted in plants with greater shootfresh weights than the three treatments containingS. alba. As with shoot lengths, the three seed mealmixtures had intermediate shoot fresh weights.

Lettuce seed viability. In the control, germinationvaried from 72% to 90%, with no significant differencesamong the treatment times (Table 2). At week 0, 0.5%S. alba seed meal was not phytotoxic and resulted ingermination rates similar to that of the control. How-ever, all mustard seed meal treatments containing

B. juncea prevented or significantly reduced lettuce seedgermination compared to the control and to 0.5% S. albaseed meal at week 0. The seed meal mixtures with thetwo highest rates of B. juncea (0.25 and 0.375%) resultedin 0% germination at week 0, and amendment with 0.2%B. juncea resulted in only 5% lettuce seed germination.During weeks 1 through 5, seed germination was notaffected by any treatment; mustard seed meal treat-ments had germination rates of 65% - 86%, all being nodifferent from the control.

Lettuce hypocotyl and root lengths. In general, let-tuce seed hypocotyl lengths were more affected by theseed meals than were root lengths (Figs. 2A, 2B). Atweek 0, all of the seed meals that did not prevent ger-mination resulted in hypocotyl lengths shorter than inthe control. The only treatment that resulted in hypo-cotyl lengths similar to the control was 0.2% B. juncea atweeks 1, 4, and 5. Although 0.5% S. alba did not reducegermination, it inhibited hypocotyl growth at all timeperiods. Effects of seed meals on root lengths weresimilar to those observed for germination (Fig. 2B). Ofthe seeds that germinated, root lengths at week 0 werelowest with 0.2% B. juncea seed meal and with 0.375%S. alba + 0.125% B. juncea. After week 0, there was little

FIG. 1. Mean shoot lengths (A) and root fresh weights (B) of pepper seedlings transplanted into soil amended with seed meals of Sinapisalba, Brassica juncea, and combinations of these meals. Amended and nonamended soils were placed into pots in the greenhouse 0 to 5 weeksprior to pepper transplanting. Application rates are percentage dry weight seed meal/dry weight soil. Shown are the means of two trials with fivereplicates of each treatment per trial (n = 10) ± standard error. Values followed by the same letter are not significantly different (P < 0.05)according to Tukey’s adjustment for multiple comparisons. Shoot lengths and root fresh weights were log10(x+1)-transformed prior to analysis;nontransformed data is presented.


significant reduction with any seed meal compared tothe control, the exceptions being 0.2% B. juncea seedmeal at week 1 and 0.25% S. alba + 0.25% B. juncea atweek 2.

Suppression of M. incognita on pepper by seed meals: Shootand root growth. Trials 1 and 2 could not be combined

due to the significant interaction between trial 3

treatment (P < 0.05) for the recorded variables. In Trials1 and 2, pepper shoots were consistently long with 0.15and 0.2% B. juncea seed meal, and with 0.05% S. alba +0.15% B. juncea (Table 3). The 0.2% S. alba and 0.15%S. alba + 0.05% B. juncea seed meals resulted in the

FIG. 2. Mean hypocotyl lengths (A) and root lengths (B) of germinating lettuce seeds exposed to soil amended with seed meals of Sinapisalba, Brassica juncea, and combinations of these meals. Amended and nonamended soils were placed into pots in the greenhouse 0 to 5 weeksprior to lettuce seed germination in Petri dishes. Application rates are percentage dry weight seed meal/dry weight soil. Shown are the means oftwo trials with thirty replicates of each treatment per trial (n = 60) ± standard error. Values followed by the same letter are not significantlydifferent (P < 0.05) according to Tukey’s adjustment for multiple comparisons.

TABLE 3. The effects of Sinapis alba and Brassica juncea seed meal soil amendments on pepper shoot lengths and weights and on root gallingindices.

Mean shoot length (cm)b Mean shoot fresh weight (g) Mean Root Galling index

Rates (% w/w)a Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Nonamended, -M. incognita 15.3 abc 17.5 abcd 2.6 cd 3.2 c 0.0 d 0.0 e0.2% S. alba 11.2 c 19.8 abc 2.2 d 4.4 ab 2.9 cd 5.8 bcd0.2% B. juncea 18.2 a 21.3 a 4.1 ab 4.8 a 1.3 d 2.3 cde0.15% S. alba 13.7 abc 20.8 ab 2.7 cd 4.8 a 5.9 bc 11.2 a0.15% B. juncea 17.2 ab 20.1 ab 3.6 abc 4.7 a 1.8 d 5.1 bcd0.1% S. alba 13.9 abc 15.6 d 3.1 abcd 3.6 bc 9.4 ab 7.0 abc0.1% B. juncea 15.8 abc 18.8 abcd 3.1 abcd 3.6 bc 9.4 ab 4.0 bcde0.05% S. alba 15.0 abc 17.1 bcd 2.8 bcd 2.9 c 7.5 ab 6.9 abc0.05% B. juncea 15.5 abc 17.6 abcd 2.9 bcd 3.4 c 6.9 ab 6.9 abc0.1% S. alba +0.1% B. juncea 16.1 abc 20.3 abc 3.4 abcd 4.6 a 1.6 d 1.9 de0.15% S. alba +0.05% B. juncea 12.5 bc 19.0 abcd 2.4 cd 4.4 ab 1.1 d 5.1 bcd0.05% S. alba +0.15% B. juncea 18.6 a 20.4 abc 4.4 a 4.5 a 1.0 d 1.9 deNonamended, +M. incognita 15.8 abc 16.4 cd 2.8 bcd 2.8 c 10.0 a 7.5 ab

aAmended and nonamended soils were inoculated with 5,000 Meloidogyne incognita eggs and placed into pots in the greenhouse 2 weeks prior to seedlingtransplant.

bValues are the mean of eight replicates of each treatment per trial (n = 8); the two trials were not combined for analysis. Within a column, values followed by thesame letter are not significantly different (P < 0.05) according to Tukey’s adjustment for multiple comparisons. Significance letters are not comparable among trialsor among columns.


shortest shoots in Trial 1, but had longer shoots in Trial2. In Trial 2, 0.1% S. alba seed meal resulted in shortshoots, along with M. incognita-inoculated plants thatwere not amended with any meal. Shoot fresh weightsalso showed differences between the two trials, but0.2% B. juncea and 0.05% S. alba + 0.15% B. juncea hadconsistently high shoot fresh weights in both trials,while 0.05% S. alba and nonamended M. incognita-inoculated plants had consistently low shoot freshweights. The two seed meals that consistently resultedin high shoot lengths and shoot fresh weights in bothtrials were 0.2% B. juncea and 0.05% S. alba + 0.15% B.juncea (Table 3). The three seed meals with consistentlyhigh root fresh weights in both trials were 0.15% and0.2% B. juncea, and 0.05% S. alba + 0.15% B. juncea(data not shown). However, there was no treatment thatresulted in a consistent, significant increase or decreasein root weight in both trials.

Root gall index and egg numbers. Trials 1 and 2could not be combined due to the significant in-teraction between trial 3 treatment (P < 0.05) for therecorded variables. In Trial 1, the root galling index washighest in the control + M. incognita, and also high withindividual low seed meal rates of 0.1% and 0.05%(Table 3). The higher rates of B. juncea (0.2 and 0.15%)and the mustard seed meal combinations resulted inthe lowest root gall indices in Trial 1. Along with highgall indices, the control + M. incognita also had the

highest number of eggs/g root in Trial 1 (Fig. 3). Thenumber of eggs/g root was lowest in the treatments thathad low gall indices; i.e. the two highest rates ofB. juncea (0.2 and 0.15%), and all three seed mealcombination amendments.

In Trial 2, the root galling index was greatest with0.15% S. alba seed meal, followed by the control +M. incognita (Table 3). In both trials, gall indices werelowest with 0.2% B. juncea, 0.1% S. alba + 0.1% B. juncea,and 0.05% S. alba + 0.15% B. juncea seed meals (Table3). Control + M. incognita, 0.1% S. alba, 0.05% S. alba,and 0.05% B. juncea consistently resulted in the highestgall indices.

In Trial 2, the numbers of eggs/g root showed overallsimilar treatment effects to Trial 1 (Fig. 3). The primaryexception was that in Trial 2, 0.1% S. alba seed meal hadmore eggs/g root than all treatments except 0.15%S. alba or the control. Lowest egg numbers/g root againoccurred in most treatments containing B. juncea seedmeal.

DISCUSSION

In our phytotoxicity study with pepper seedlings andlettuce seeds, S. alba seed meal was applied at a higherrate than B. juncea seed meal, because the latter haddemonstrated greater nematotoxicity (Zasada et al.,2009), and our ultimate goal was to suppress nematode

FIG. 3. Effects of Sinapis alba (Sa) and Brassica juncea (Bj) seed meals applied alone and in combinations on numbers of Meloidogyne incognita(root-knot nematode) eggs/g root on greenhouse-grown pepper plants. Amended and nonamended soils were inoculated with M. incognitaeggs (5,000 per pot) and placed into pots 2 weeks prior to pepper seedling transplant. Application rates are percentage dry weight seed meal:dryweight soil. Shown are the means of eight replicates of each treatment (n = 8) per trial; the two trials were not combined for analysis. Valuesfollowed by the same letter are not significantly different (P < 0.05) within a trial according to Tukey’s adjustment for multiple comparisons;significance values are not comparable between trials.


populations. For example, an individual applicationrate of 0.5% B. juncea and S. alba seed meals resulted inM. incognita suppression of 100% and ca. 80%, re-spectively (Zasada et al., 2009). We also chose to applymeals with a reduced flake size, because a flake appli-cation of S. alba seed meal, particularly smaller flakes,improved activity against P. penetrans (Zasada et al.,2009). The improved efficacy against pathogens withdecreased particle size is most likely a result of higherisothiocyanate generation with small particles (Mazzolaand Zhao, 2010). With these results in mind, thenematotoxic rates of 0.5% S. alba and 0.2% B. juncea,applied as ground seed meals, were selected for in-dividual meal applications in our phytotoxicity tests.The 0.5% S. alba amendment was more phytotoxic topepper seedlings than the 0.2% B. juncea seed mealamendment, resulting in more pepper plant death andin smaller shoots and roots than the B. juncea seedmeal. The seed meal combinations tended to be in-termediate in effect on pepper seedlings, althoughroots were small in all seed meals except 0.2% B. juncea.This study indicated that, based on the recorded pa-rameters, only the 0.2% B. juncea amendment could beused without some phytotoxic effects within weeks ofpepper seedling transplant, with the earliest applica-tion time being 2 weeks prior to transplant.

The response of lettuce seeds to seed meals was dif-ferent from that of pepper seedlings. Immediately afterapplication (week 0), 0.5% S. alba did not inhibit let-tuce seed germination, but all treatments containingB. juncea did. This toxic effect of B. juncea was gone byweek 1. Sinapis alba seed meal did decrease hypocotyllengths compared to the control at all times, althoughroot length was unaffected. Brassica juncea seed mealdecreased hypocotyl length to some extent the first fewweeks, and root lengths weeks 0 and 1.

The differential effects of the seed meals in thesephytotoxicity tests may have been due to several factors,including seed meal species, application rates, plantspecies, and seedling growth vs. seed germination.Mustard plant species differ in types and amounts ofglucosinolates, and therefore in breakdown productssuch as isothiocyanates (ITC), ionic thiocyanate(SCN-), nitriles, and oxazolidinethiones (Borek andMorra, 2005; Brown and Morra, 2005; Hansson et al.,2008). Both types of seed meals tested are known tohave high glucosinolate contents; B. juncea ‘PacificGold’ mainly contains sinigrin (2-propenyl glucosino-late), while S. alba ‘‘IdaGold’ contains a large concen-tration of sinalbin (4-hydroxybenzyl glucosinolate)(Borek and Morra, 2005; Rice et al., 2007; Hanssonet al., 2008). Typical concentrations of sinigrin in ‘Pa-cific Gold’ seed meal range from 108-134 mmol/g, andtypical sinalbin concentrations in ‘IdaGold’ meal rangefrom 125-160 mmol/g (Morra, unpublished). Theglucosinolates in S. alba form SCN- in soil, which isknown to act as an herbicide (Borek and Morra, 2005);

glucosinolates in B. juncea seed meal produce ITCs thatare also phytotoxic (Rice et al., 2007). Also, whileB. juncea produces compounds that are toxic on con-tact, so the plant is either tolerant or dies quickly, SCN-

is translocated and accumulates in plant tissues (Stiehland Bible, 1989; Brown and Morra, 2005). An exampleof disparate activity of these two seed meals on thesame crop plant species was shown with carrot; S. albaamendment inhibited emergence compared with B.juncea (Hansson et al., 2008; Snyder et al., 2009). Inaddition, the seed meal application rates used in ourstudy were selected for nematotoxicity, so S. alba wasapplied at a higher rate, which could also cause somedifferences in phytotoxicity between seed meals.

The dissimilar results observed on pepper comparedwith lettuce may also be due to differential sensitivity toglucosinolate breakdown products that have been ob-served among plant species (Vaughn et al., 2006). Forexample, when seeds and seedlings of 39 crop plantspecies were exposed to SCN-, 44% of the crop plantstested did not exhibit adverse effects (Stiehl and Bible,1989). Not all plant species are equally sensitive to thephytotoxic chemicals. Finally, another factor that couldaffect results with pepper seedlings vs. lettuce seeds isthat even when percentage seed germination is notinhibited by SCN-, subsequent plant growth can still beinhibited by this compound (Stiehl and Bible, 1989;Brown and Morra, 2005). Similarly, with B. juncea seedmeal, phytotoxicity was observed with sweet corn, butgermination was not inhibited (Yu et al., 2007).

In the M. incognita suppression studies with pepper,S. alba and B. juncea seed meal application rates werebased on the phytotoxicity test results. The two seedmeals were applied at equal rates, and as 1:1 and 1:3combinations. The three seed meal application ratesthat resulted in the longest pepper shoots and thegreatest shoot and root weights were 0.2% B. juncea,0.15% B. juncea and the low S. alba + high B. juncea(0.05% + 0.15%) combination. Of these three seedmeal amendments, 0.2% B. juncea and 0.05% S. alba +0.15% B. juncea also were among the lowest in rootgalling indices; 0.15% B. juncea varied between trials.The 0.1% S. alba + 0.1% B. juncea combination alsotended to have high shoot and root growth and low gallindices. In both trials, when similar rates of S. albaand B. juncea were applied as individual seed meals,B. juncea generally had a more suppressive effect onM. incognita populations than S. alba, except at the lowestrate (0.05%). These results agree with previous studiesindicating that B. juncea seed meal is more active than S.alba against P. penetrans and M. incognita (Mazzola et al.,2007, 2009; Zasada et al., 2009). Brassica juncea seedmeal also suppressed M. javanica populations in vine-yards (Rahman and Somers, 2005), Tylenchulus semi-penetrans in soil (Walker, 1997), and P. penetrans onsweet corn (Yu et al., 2007). This activity may be at leastin part due to high levels of sinigrin. Seed meal made


from Brassica carinata, which is also high in this com-pound, was efficacious for suppressing M. incognita onzucchini in a commercial greenhouse (Lazzeri et al.,2009).

The seed meal combinations tested in our study alsotended to be suppressive to M. incognita. The range ofseed meals available provides for the possibility of pro-ducing combinations that are active against multipleplant pests or pathogens. Soil application of a 1:1 ratioof B. juncea to B. napus seed meal improved apple re-plant disease control, partly because of differential ef-fects of each meal on plant-pathogenic fungi (Mazzolaand Brown, 2010). Consequently, a combination of S.alba and B. juncea seed meals active against M. incognitamight have additional benefits for suppression of weedsor of other pathogens.

The results of this study indicate that the lower phy-totoxicity of B. juncea seed meal to pepper, combinedwith the greater nematotoxicity of this seed meal, makeit a better candidate than S. alba when used alone forsuppression of M. incognita on pepper. However, seedmeal amendments containing a higher rate of B. junceacombined with a lower rate of S. alba could also beformulated for concurrent nematode and weed sup-pression. Further studies would indicate whether both ofthese pests can indeed be minimized with such a com-bination. Additional tests can also determine whetherlong-term growth of pepper in amended, unpasteurizedsoil could allow for enhanced microbial activity, possiblyresulting in even greater suppressive activity.

Acknowledgments. Thanks are extended to ShannonRupprecht for greenhouse and laboratory work and dataanalysis, to Ashlee Green and Emily Brinker for assistancein the greenhouse and laboratory, and to Vladimir Borekfor providing mustard seed meal. Dr. Orisajo’s visit to theNematology Laboratory was supported by a Norman E.Borlaug International Agricultural Science and Technol-ogy Fellows Program grant from the World Cocoa Foun-dation and the USDA.

LITERATURE CITED

Borek, V., and Morra, M. J. 2005. Ionic thiocyanate (SCN-) pro-duction from 4-hydroxybenzyl glucosinolate contained in Sinapis albaseed meal. Journal of Agricultural and Food Chemistry 53:8650–8654.

Boydston, R. A., Anderson, T., and Vaughn, S. F. 2008. Mustard(Sinapis alba) seed meal suppresses weeds in container-grown orna-mentals. HortScience 43:800–803.

Brown, P. D., and Morra, M. J. 1995. Glucosinolate-containing planttissues as bioherbicides. Journal of Agricultural and Food Chemistry43:3070–3074.

Brown, J., and Morra, M. J. 2005. Glucosinolate-containing seedmeal as a soil amendment to control plant pests 2000-2002. NationalRenewable Energy Laboratory Subcontract Report NREL/SR-510-35254 95 pp.

Buskov, S., Serra, B., Rosa, E., Sorensen, H., and Sorensen, J. C.2002. Effects of intact glucosinolates and products produced fromglucosinolates in myrosinase-catalyzed hydrolysis on the potato cyst

nematode (Globodera rostochiensis cv. Woll). Journal of Agricultural andFood Chemistry 50:690–695.

Cohen, M. F., and Mazzola, M. 2004. A reason to be optimistic aboutbiodiesel: seed meal as a valuable soil amendment. Trends in Bio-technology 22:211–212.

Cohen, M. F., and Mazzola, M. 2006. Resident bacteria, nitric oxideemission and particle size modulate the effect of Brassica napus seedmeal on disease incited by Rhizoctonia solani and Pythium spp. Plantand Soil 286:75–86.

Cohen, M. F., Yamasaki, H., and Mazzola, M. 2005. Brassica napusseed meal soil amendment modifies microbial community structure,nitric oxide production and incidence of Rhizoctonia root rot. SoilBiology & Biochemistry 37:1215–1227.

Cox, C. J., McCarty, L. B., Toler, J. E., Lewis, S. A., and Martin, S. B.2006. Suppressing sting nematodes with Brassica sp., poinsettia, andspotted spurge extracts. Agronomy Journal 98:962–967.

Daulton, R. A. C. 1959. Soil temperature and soil moisture factorsaffecting the survival of eggs of the root-knot nematodes Meloidogynejavanica javanica and M. hapla. PhD dissertation, North Carolina StateCollege, Raleigh, NC.

Donkin, S. G., Eiteman, M. A., and Williams, P. L. 1995. Toxicity ofglucosinolates and their enzymatic decomposition products to Cae-norhabditis elegans. Journal of Nematology 27:258–262.

Hansson, D., Morra, M. J., Borek, V., Snyder, A. J., Johnson-Maynard, J. L., and Thill, D. C. 2008. Ionic thiocyanate (SCN-) pro-duction, fate, and phytotoxicity in soil amended with Brassicaceaeseed meals. Journal of Agricultural and Food Chemistry 56:3912–3917.

Henderson, D. R., Riga, E., Ramirez, R. A., Wilson, J., andSynder, W. E. 2009. Mustard biofumigation disrupts biological controlby Steinernema spp. nematodes in the soil. Biological Control 48:316–322.

Hoagland, L., Carpenter-Boggs, L., Reganold, J. P., and Mazzola, M.2008. Role of native soil biology in brassicaceous seed meal-inducedweed suppression. Soil Biology & Biochemistry 40:1689–1697.

Lazzeri, L., Curto, G., Dallavalle, E., D’Avino, L., Malaguti, L.,Santi, R., and Patalano, G. 2009. Nematicidal efficacy of bio-fumigation by defatted Brassicaceae meal for control of Meloidogyneincognita (Kofoid et White) Chitw. on a full field zucchini crop.Journal of Sustainable Agriculture 33:349–358.

Lazzeri, L., Curto, G., Leoni, O., and Dallavalle, E. 2004. Effects ofglucosinolates and their enzymatic hydrolysis products via myrosinaseon the root-knot nematode Meloidogyne incognita (Kofoid et White)Chitw. Journal of Agricultural and Food Chemistry 52:6703–6707.

Lazzeri, L., Tacconi, R., and Palmieri, S. 1993. In vitro activity ofsome glucosinolates and their reaction products toward a populationof the nematode Heterodera schachtii. Journal of Agricultural and FoodChemistry 41:825–829.

Mazzola, M., and Brown, J. 2010. Efficacy of brassicaceous seed mealformulations for the control of apple replant disease in conventionaland organic production systems. Plant Disease 94:835–842.

Mazzola, M., Brown, J., Izzo, A. D., and Cohen, M. F. 2007. Mech-anism of action and efficacy of seed meal-induced pathogen sup-pression differ in a Brassicaceae species and time-dependent manner.Phytopathology 97:454–460.

Mazzola, M., Brown, J., Zhao, X., Izzo, A. D., and Fazio, G. 2009.Interaction of brassicaceous seed meal and apple rootstock on re-covery of Pythium spp. and Pratylenchus penetrans from roots grown inreplant soils. Plant Disease 93:51–57.

Mazzola, M., Granatstein, D. M., Elfving, D. C., and Mullinix, K.2001. Suppression of specific apple root pathogens by Brassica napusseed meal amendment regardless of glucosinolate content. Phytopa-thology 91:673–679.

Mazzola, M., and Mullinix, K. 2005. Comparative field efficacy ofmanagement strategies containing Brassica napus seed meal or greenmanure for the control of apple replant disease. Plant Disease89:1207–1213.


Mazzola, M., and Zhao, X. 2010. Brassica juncea seed meal particlesize influences chemistry but not soil biology-based suppression ofindividual agents inciting apple replant disease. Plant and Soil: publishedonline http://www.springerlink.com/content/p3852010w8214k87/fulltext.pdf.

Meyer, S. L. F., Lakshman, D. K., Zasada, I. A., Vinyard, B. T., andChitwood, D. J. 2008. Phytotoxicity of clove oil to vegetable cropseedlings and nematotoxicity to root-knot nematodes. HortTechnol-ogy 18:631–638.

Rahman, L., and Somers, T. 2005. Suppression of root knot nem-atode (Meloidogyne javanica) after incorporation of Indian mustard cv.Nemfix as green manure and seed meal in vineyards. AustralasianPlant Pathology 34:77–83.

Rice, A. R., Johnson-Maynard, J. L., Thill, D. C., and Morra, M. J.2007. Vegetable crop emergence and weed control followingamendment with different Brassicaceae seed meals. Renewable Agri-culture and Food Systems 22:204–212.

Snyder, A., Morra, M. J., Johnson-Maynard, J., and Thill, D. C. 2009.Seed meals from Brassicaceae oilseed crops as soil amendments: in-fluence on carrot growth, microbial biomass nitrogen, and nitrogenmineralization. HortScience 44:354–361.

Stiehl, B., and Bible, B. B. 1989. Reaction of crop species to thio-cyanate ion toxicity. HortScience 24:99–101.

Vaughn, S. F., Palmquist, D. E., Duval, S. M., and Berhow, M. A.2006. Herbicidal activity of glucosinolate-containing seedmeals. WeedScience 54:743–738.

Walker, G. E. 1997. Effects of Brassica residues and other organicamendments on abundance and sex ratio of Tylenchulus semipenetransin soil. Australian Journal of Experimental Agriculture 37:693–700.

Walters, T. W., Pinkerton, J. N., Riga, E., Zasada, I. A., Particka, M.,Yoshida, H. A., and Ishida, C. 2009. Managing plant-parasitic nema-todes in established red raspberry fields. HortTechnology 19:762–768.

Yu, Q., Tsao, R., Chiba, M., and Potter, J. 2005. Selective nematicidalactivity of allyl isothiocyanate. Journal of Food, Agriculture and En-vironment 3:218–221.

Yu, Q., Tsao, R., Chiba, M., and Potter, J. 2007. Elucidation of thenematicidal activity of bran and seed meal of oriental mustard(Brassica juncea) under controlled conditions. Journal of Food, Agri-culture & Environment 5:374–379.

Zasada, I. A., and Ferris, H. 2003. Sensitivity of Meloidogyne javanicaand Tylenchulus semipenetrans to isothiocyanates in laboratory assays.Phytopathology 93:747–750.

Zasada, I. A., Meyer, S. L. F., and Morra, M. J. 2009. Brassicaceousseed meals as soil amendments to suppress the plant-parasitic nem-atodes Pratylenchus penetrans and Meloidogyne incognita. Journal ofNematology 41:221–227.


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The Society of Nematologists 2011. Mustard seed meal …€¦ · Mustard seed meal mixtures: management of Meloidogyne incognita on pepper and potential phytotoxicity SUSAN L. F